DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca²⁺-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T₀ regenerants and sprdr6 null T₁ progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

A DNA-free CRISPR-Cas9 genome editing tool based on an optimized protoplast regeneration protocol of wild tomato creates stable and inheritable diploid and tetraploid regenerants.     

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion–associated protein Zyxin to regulate UV-induced cell death

Zyxin is a zinc-binding phosphoprotein known to regulate cell migration, adhesion, and cell survival. Zyxin also plays a role in signal transduction between focal adhesions and the nuclear compartment. However, the mechanism of Zyxin shuttling to nucleus is still unclear. Here, we identify that the GlcNAc transferase (O-linked GlcNAc [O-GlcNAc] transferase) can O-GlcNAcylate Zyxin and regulate its nuclear localization. We show that O-GlcNAc transferase O-GlcNAcylates Zyxin at two residues, serine 169 (Ser-169) and Ser-246. In addition, O-GlcNAcylation of Ser-169, but not Ser-246, enhances its interaction with 14-3-3γ, which is a phosphoserine/threonine-binding protein and is reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3γ could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export signal sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that the nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death.     

G-quadruplex inducer/stabilizer pyridostatin targets SUB1 to promote cytotoxicity of a transplatinum complex

Pyridostatin (PDS) is a well-known G-quadruplex (G4) inducer and stabilizer, yet its target genes have remained unclear. Herein, applying MS proteomics strategy, we revealed PDS significantly downregulated 22 proteins but upregulated 16 proteins in HeLa cancer cells, of which the genes both contain a number of G4 potential sequences, implying that PDS regulation on gene expression is far more complicated than inducing/stabilizing G4 structures. The PDS-downregulated proteins consequently upregulated 6 proteins to activate cyclin and cell cycle regulation, suggesting that PDS itself is not a potential anticancer agent, at least toward HeLa cancer cells. Importantly, SUB1, which encodes human positive cofactor and DNA lesion sensor PC4, was downregulated by 4.76-fold. Further studies demonstrated that the downregulation of PC4 dramatically promoted the cytotoxicity of trans-[PtCl₂(NH₃)(thiazole)] (trans-PtTz) toward HeLa cells to a similar level of cisplatin, contributable to retarding the repair of 1,3-trans-PtTz crosslinked DNA lesion mediated by PC4. These findings not only provide new insights into better understanding on the biological functions of PDS but also implicate a strategy for the rational design of novel multi-targeting platinum anticancer drugs via conjugation of PDS as a ligand to the coordination scaffold of transplatin for battling drug resistance to cisplatin.     

Acyl-CoA synthetase long-chain 3-mediated fatty acid oxidation is required for TGFβ1-induced epithelial-mesenchymal transition and metastasis of colorectal carcinoma

Cancer cells frequently undergo metabolic reprogramming to support tumorigenicity and malignancy, which is recognized as a hallmark of cancer. In addition to glycolysis and glutaminolysis, alterations in fatty acid (FA) metabolism have received increasing concerns in the past few years. Recently, accumulating evidence has shown that fatty acid β-oxidation (FAO) is abnormally activated in various tumors, which is associated with the machinery of proliferation, stemness, metastasis, and radiochemotherapeutic resistance of cancer cells. Acyl-CoA synthetases 3 (ACSL3) belongs to a family of enzymes responsible for converting free long-chain FAs into fatty acyl-CoA esters, which act as substrates both for lipid synthesis and FAO.Here, we demonstrate that transforming growth factor beta 1 (TGFβ1) induces the up-regulation of ACSL3 through sterol regulatory element-binding protein 1 (SREBP1) signaling to promote energy metabolic reprogramming in colorectal carcinoma (CRC) cells. ACSL3 mediates the epithelial mesenchymal transition (EMT) and metastasis of CRC cells by activation of FAO pathway to produce ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which sustain redox homeostasis and fuel cancer cells for invasion and distal metastasis. Thus, targeting ACSL3 and FAO metabolic pathways might be exploited for therapeutic gain for CRC and other FAs- addicted cancers.     

iASPP protects the heart from ischemia injury by inhibiting p53 expression and cardiomyocyte apoptosis

Currently, there remains a great need to elucidate the molecular mechanism of acute myocardial infarction in order to facilitate the development of novel therapy. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a member of the ASPP family proteins and an evolutionarily preserved inhibitor of p53 that is involved in many cellular processes, including apoptosis of cancer cells. The purpose of this study was to investigate the possible role of iASPP in acute myocardial infarction. The protein level of iASPP was markedly reduced in the ischemic hearts in vivo and hydrogen peroxide-exposed cardiomyocytes in vitro. Overexpression of iASPP reduced the infarct size and cardiomyocyte apoptosis of mice subjected to 24 h of coronary artery ligation. Echocardiography showed that cardiac function was improved as indicated by the increase in ejection fraction and fractional shortening. In contrast, knockdown of iASPP exacerbated cardiac injury as manifested by impaired cardiac function, increased infarct size, and apoptosis rate. Mechanistically, overexpression of iASPP inhibited, while knockdown of iASPP increased the expressions of p53 and Bax, the key regulators of apoptosis. Taken together, our results suggested that iASPP is an important regulator of cardiomyocyte apoptosis, which represents a potential target in the therapy of myocardial infarction.     

SorCS3 promotes the internalization of p75NTR to inhibit GBM progression

Glioblastoma (GBM) is a fatal malignancy caused by dysregulation of cellular signal transduction. Internalization plays a key role in maintaining signalling balance. Previous reports showed that Sortilin related VPS10 domain containing receptor 3 (SorCS3) has the ability to regulate internalization. However, the impacts of SorCS3 on the biological processes involved in GBM have not yet been reported. In this study, we investigated the bio-function of SorCS3 in GBM. We found that SorCS3 was significantly downregulated in GBM. In addition, low expression level of SorCS3 predicted poor prognoses in patients with GBM. Here, we proved that SorCS3 suppressed cell invasion and proliferation mainly via NGF/p75^NTR pathway in GBM. We found that SorCS3 co-localized with p75^NTR in GBM cells and regulated the p75^NTR protein level by promoting trafficking of the endosomal to the lysosome. Immunofluorescence (IF) and Co-Immunoprecipitation (Co-IP) detection confirmed that SorCS3 bound to p75^NTR, which subsequently increased the internalization of p75^NTR, and then transported p75^NTR to the lysosome for degradation, ultimately contributing to inhibit of glioma progression. Taken together, our work suggests that SorCS3 is a marker of promising prognosis in GBM patients and suggests that SorCS3 regulates internalization, which plays a pivotal role in inhibiting glioma progression.Subject terms: Tumour-suppressor proteins, Protein transport     

应用iTRAQ技术分析串子花型滩羊初生期与二毛期的差异蛋白

为了解二毛期时串子花型滩羊羊毛弯曲形成机理,本试验采用iTRAQ技术及LC-MS/MS的研究方法,对初生期和二毛期串子花型滩羊的皮肤样品进行蛋白质鉴定和筛选,并运用Proteome Discoverer 1.4软件进行定量分析,结合数据库搜索,鉴定出具有显著表达差异的蛋白,同时应用生物学技术对其进行GO和KEGG Pa...     

中国不同地域恒河猴MHC-Ⅰ型部分等位基因的调查

采用序列特异性引物-聚合酶链式反应(PCR-SSP)分型方法对在华南灵长类动物研究中心繁殖的247只中国恒河猴(Macaca mulatta)(其中30只来源于广西、34只来源于海南,183只来源于川西、安徽等内陆地区杂交群)的Mamu-A01、A02、A08、B01和NA7等5个MHC-Ⅰ型分子位点进行检测。结果显示,A01、A02发现于海南群和杂交群,阳性率均小于8.8%;A08发现于杂交群中,阳性率小于3.8%;B01发现于广西群和杂交群,阳性率均大于17.48%;NA7在3个猴群中均有发现,阳性率均大于32.4%。中国不同地域恒河猴群体携带的5个MHC-Ⅰ型等位基因的频率存在明显差异,通过与印度的恒河猴比较,中国恒河猴与印度恒河猴携带的MHC-Ⅰ等位基因也存在显著的差异。     

松萝内生酿酒酵母菌株耐酸生理特性与分子机制探究

【目的】对我国西藏地区来源的不同酵母菌株进行有机酸发酵性能测试,此外,对具有良好产酸性能的分离自松萝内部的酿酒酵母菌株Saccharomy cescerevisiae 2-2进行耐酸性能分析,并探究其耐酸较强的分子机制。【方法】比较不同糖浓度培养基液体发酵培养过程中pH的变化,并比较低pH胁迫条件下菌株的生长,检测酿酒酵母菌株的产酸潜力和耐酸特性;对菌株2-2和模式酵母菌株S288C进行比较基因组分析,并利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)分析关键基因的转录,探究菌株2-2耐酸分子机制。【结果】松萝内生酿酒酵母2-2在所有检测的菌株中产酸潜力较大,耐酸性能较好。在菌株2-2中与胁迫耐受性相关的基因PDR15、PDR12和SUR1在低pH胁迫条件下存在显著的上调或下调,但这些基因转录变化趋势与菌株S288C相反。【结论】松萝内生酿酒酵母2-2是一株产酸耐酸性能较好的菌株,对其独特的调节机制进行深入分析,有希望选育性能更好的产酸酵母菌株。